The invention relates to fluorescent protein sensors for detecting and quantifying analytes.
Measurement of an analyte concentration in vitro or in vivo by non-invasive techniques can help elucidate the physiological function of the analyte. This can also aid in identifying changes that occur in a cell or organism in response to physiological stimuli. For example, cyclic AMP can be detected by fluorescence resonance energy transfer between a separately labeled proteins that associate with each other but are not covalently attached to each other. See, U.S. Pat. No. 5,439,797.
For example, many effects of Ca2+ in cells are mediated by Ca2+ binding to calmodulin (CaM), which causes CaM to bind and activate target proteins or peptide sequences. Based on the NMR solution structure of CaM bound to the 26-residue M13 Ca2+-binding peptide of myosin light-chain kinase, Porumb et al. fused the C-terminus of CaM via a Gly-Gly spacer to the M13. Ca2+ binding switches the resulting hybrid protein (CaM-M13) from a dumbbell-like extended form to a compact globular form similar to the CaM-M13 intermolecular complex. See, Porumb, T., et al., Prot. Engineering 7:109-115 (1994).
Fluorescent Ca2+ indicators such as fura-2, indo-1, fluo-3, and Calcium-Green have been the mainstay of intracellular Ca2+ measurement and imaging. See, for example, U.S. Pat. No. 4,603,209 and U.S. Pat. No. 5,049,673. These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents. Although the Ca2+ indicating photoprotein aequorin is targetable, the photoresponse to Ca2+ is low since it is chemiluminescent. Moreover, aequorins need to incorporate exogenous coelenterazine.